For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contactHumana at the above address or at any of the following numbers: Tel.: 973-256-1699; Fax: 973-2568341; E-mail: humana@humanapr.com or visit our website: http://humanapress.comThis publication is printed on acid-free paper. ∞ANSI Z39.48-1984 (American National Standards Institute) Permanence of Paper for Printed LibraryMaterials.Photocopy Authorization Policy:Authorization to photocopy items for internal or personal use, or the internal or personal use of specificclients, is granted by Humana Press Inc., provided that the base fee of US $20.00 per copy is paid directlyto the Copyright Clearance Center at 222 Rosewood Drive, Danvers, MA 01923. For those organizationsthat have been granted a photocopy license from the CCC, a separate system of payment has beenarranged and is acceptable to Humana Press Inc. The fee code for users of the Transactional ReportingService is: [0-89603-815-7/03 $20.00].Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1Library of Congress Cataloging-in-Publication DataMethods in biological oxidative stress / edited by Kenneth Hensley, Robert A. Floyd.p. cm.

Oxidative damage appears to play a central role in the development ofa wide range of tissue pathology, including neurodegenerative disease, drugside-effects, xenobiotic toxicity, carcinogenesis, and the aging process,to name just a few.Because of the centrality of oxidative processes to normal and abnormaltissue function, it has become imperative to develop appropriate analyticaltechniques to facilitate the quantitation of significant reactants. Withoutadvances in methodology, corresponding advances in our knowledge ofunderlying biochemical events will be necessarily limited.Drs. Hensley and Floyd have done an outstanding job of assembling thework of world-class experts into Methods in Biological Oxidative Stress.The contributors have presented concise, yet thorough, descriptions of thestate-of-the-art methods that any investigator working in the field needs toaccess.Mannfred A. Hollinger

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PrefaceFree radicals and reactive oxidizing agents were once ignored asbiochemical entities not worth close scrutiny, but are now recognized ascauses or contributing factors in dozens, if not hundreds, of disease states. Inaddition, free radical metabolisms of xenobiotics have become increasinglyimportant to pharmacologists. Accordingly, the need has arisen to accuratelyquantify reactive oxygen species and their byproducts.Methods in Biological Oxidative Stress is practical in scope, providingthe details of up-to-date techniques for measuring oxidative stress anddetecting oxidizing agents both in vitro and in vivo. The contributors arerecognized experts in the field of oxidative stress who have developed novelstrategies for studying biological oxidations.The chapters of Methods in Biological Oxidative Stress cover widely usedstandard laboratory techniques, often developed by the authors, as well asHPLC–electrochemical measurement of protein oxidation products, particularlynitrotyrosine and dityrosine, and HPLC–electrochemical detection of DNAoxidation products. Additionally, recently developed techniques arepresented to measure lipid oxidation and nitration products such as 5-NO2γ-tocopherol and isoprostanes, using HPLC-electrochemical/photodiodearray methods and mass spectrometry as well as electron paramagneticresonance (EPR) techniques.In scope, presentation, and authority therefore, Methods in BiologicalOxidative Stress was designed to be an invaluable manual for clinicallaboratories and teaching institutions now conducting routine measurementsof biological oxidants and biological oxidative stress or implementing newprograms in this vital area of research. As a reference work, this collectionof techniques and methods will prove useful for many years to come.Kenneth HensleyRobert A. Floyd

cal detection (ECD) is typically chosen for its enhanced selectivity and sensitivity, especially when trying to measure low levels of analytes (e.g., K1,CoQ10) in low volume-low level samples (e.g., fasting or neonatal plasma).Single- and dual-channel ECDs are typically used at settings that are suitable for only a few analytes at the expense of others’ whereas multi-component analyses are limited by the poor compatibility of thin-layeramperometric electrodes with gradient elution chromatography (6).An alternate electrochemical approach uses a serial array of highly efficient (coulometric) flow-through graphite working electrodes maintained atdifferent but constant potentials, each optimal for a given analyte or class ofanalytes (the CoulArray® - ESA Inc.) (7,8). When combined with gradientHPLC, a three-dimensional chromatogram is generated that identifies ananalyte by both retention time and electrochemical (hydrodynamicvoltammetric) behavior. The latter, like a photodiode array spectrum, can beused to verify analyte authenticity or to identify co-eluting or misnamedanalytes. This approach is finding great use in the field of oxidative metabolism for the measurement of water- and fat-soluble antioxidants, DNAadducts, and protein oxidation products (2,3,9–11).Presented here are three methods using HPLC-coulometric array detection:1. Method 1: A global method capable of measuring vitamins A, and E as well asCoQ10, retinoids and carotenoids in plasma and serum.2. Method 2: A second global method that also includes vitamins D2 and D3 forthe analysis of milk sample.3. Method 3: A method for the measurement of carotenoid isomers in plasmaand serum.

2. MATERIALS1. The analytical system for Methods 1 and 2 consisted of a model 5600CoulArray 8-channel system with two model 582 pumps, a high pressure gradient mixer, a PEEK® pulse dampener, a model 540 autosampler, a CoulArraythermostatic chamber and a serial array of eight coulometric electrodes (allfrom ESA, Inc.) The apparatus for Method 3 was the same as the other methods, but used a single pump.2. Standards for Methods 1 and 2 were obtained from Sigma Chemical Co. (St.Louis, MO). Stock standards were made by dissolving approx 10 mg of eachcompound in 10 mL of ethanol (EtOH) with the exception of the carotenoidsand Q10. For these more lipophilic compounds, ~1.0 mg were dissolved in 5.0mL of hexane followed by dilution with 15 mL EtOH. Stock solutions were

Fig. 1. The chemical structures of some fat-soluble vitamins and antioxidants.

b. Method 2. Milk samples (unsaponified): A 1.0 mL volume, augmentedwith 10 μL of 1.0 μg/mL D2 (internal standard), was thoroughly mixedwith 3.0 mL diluent and 0.1 g magnesium sulfate. The resulting mixturewas extracted two times with 4.0 mL hexane. Combined hexane extractswere evaporated under a stream of nitrogen and residue was dissolved in1.0 mL of diluent. The solution was centrifuged as in Method 1. Milksamples (saponified): a 1.0 mL volume of milk was mixed with 1.75 mL85% aqueous EtOH containing 75 mg/mL potassium hydroxide and 0.25mg/mL ascorbic acid. The sample was then placed in a heated water bathfor 45 min at 95°C. Saponified samples were then extracted as forunsaponified milk samples.c. Method 3. A 0.5 mL volume of serum or standard was mixed with 0.5 mLethanol/10 mg/L BHA. After mixing for 1 min, 1.5 mL of hexane was addedand after mixing for an additional 10 min was centrifuged (4,000g, 10 min).Approx 1.0 mL of supernatant was withdrawn and the remaining sampleextracted with an additional 1.5 mL of hexane. Combined hexane extractswere evaporated to dryness under a stream of nitrogen. Finally, the residuewas dissolved in 0.25 mL of mobile phase.

4. RESULTS AND DISCUSSIONThe global method (Method 1) combines the resolution of gradient HPLCwith coulometric array detection to separate and identify FSVAs in under 30min [Fig. 2A, 2B; extracted standards and a typical NIST (National InstituteScience and Technology) control human serum, respectively]. The RNSmarker, 5-nitro-a-tocopherol, eluted at 31 min (data not shown) (see ref. 3).The tocopherols were the most easily oxidized and were measured on channel 1 (200 mV) of the array. The carotenoids responded on channel 2 (400mV) while the retinoids were the highest oxidizing compounds and reactedmainly on channel 4 (700 mV). Vitamin K1 (not shown) and CoQ10 onlyresponded after their reduction at –1000 mV on channel 6 followed by facileoxidation at +200 mV on channel 7.The assay had a sensitivity in the low picogram range (e.g., retinol [alltrans], _-tocopherol, and CoQ10 were 3.8, 5.1, and 7.5 pg on column,respectively) and was linear from 0–10 μg/mL. Ratio accuracies, indicatorsof analyte authenticity, were >0.850 (6,7). The levels of analytes determinedby this method correlated well with NIST published values (Table 1).The chromatography and electrochemical array conditions used inMethod 2 were optimized for a wide range of FSVAs, including vitaminsD2 and D3 (Fig. 3). The first electrode in the array was set to –700 mV toreduce vitamin K1 and CoQ10, these were then measured oxidatively onsensors 2 and 3, respectively.

Fig. 2. Gradient HPLC–coulometric array chromatograms of (A) extracted standards and (B) NIST control plasma sample.Signal from the reduction channels (6 and 7) is not included.